The present invention relates to a process for preparing cephalotaxane derivatives bearing a side chain.
The term xe2x80x9ccephalotaxanesxe2x80x9d refers to compounds or salts thereof which have a basic skeleton of formula 
where p is equal to 1 or 2 (it being possible for the two units to be identical or different and linked via a single bond or an oxygen atom), which can contain various oxygenated substituents (aliphatic or aromatic ethers, free or esterified alcohols, substituted or free enols and/or phenols, bridged ethers, and more generally any substituent usually encountered in the natural state on compounds of this type).
Harringtonines are alkaloids which are of high interest in anticancer chemotherapy, in particular on certain haematosarcomas which are multi-resistant to the existing therapies. The selectivity of harringtonines, which is based on a novel mechanism of action relating to protein synthesis, is such that this series is favoured with a great future in anticancer therapy.
Several literature compilations give a seemingly exhaustive review of all of the knowledge relating to cephalotaxanes, these compilations being, chronologically: [C. R. Smith, Jr, R. G. Powell and K. L. Mikolajczack, Cancer Treat. Rep., Vol. 60, 1157 (1976); C. R. Smith, Jr, L. Kenneth, K. L. Mikolajczack and R. G. Powell in xe2x80x9cAnticancer Agent Based on Natural Product Modelxe2x80x9d, 391 (1980); Liang Huang and Zhi Xue in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. XXIII (A. Brossi Ed.), 157 (1984); M. Suffness and G. A. Cordell in xe2x80x9cThe Alkaloids, Chemistry and Pharmacologyxe2x80x9d (A. Brossi Ed.), Vol. 25, 57-69, 295-298 (1""987); P. J. O""Dwyer, S. A. King, D. F. Hoth, M. Suffness and B. Leyland-Jones, Journal of Clinical Oncology, 1563 (1986); T. Hudlicky, L. D. Kwart and J. W. Reed, in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, 199 (1998)].
Antiparasitic activities, in particular on the haematozoon of malaria, have also been recognized [J. M. Whaun and N. D. Brown, Ann Trop. Med. Par., Vol. 84, 229 (1990)].
Homo-harringtonine (HHT), the most active member of the series, is active at and above daily doses of 2.5 mg/m2 of body area per 24 hours, i.e., as a guide, at doses twenty times lower than that for Taxol. HHT has already undergone fourteen phase I and II clinical trials and it is the only known product capable of a 70% reinduction of full haematological remissions in patients suffering from chronic myeloid leukaemias that have become resistant to alpha-interferon [S. O""Brien, H. Kantarjian, M. Keating, M. Beran, C. Koler, L. E. Robertson, J. Hester, M. Rios, M. Andreeff and M. Talpaz, Blood, 332 (1995); Leukemia Insights, Vol. 3, No. 1 (1998)].
Harringtonines were extracted over 35 years ago from an exclusively Asiatic cephalotaxacea known as Cephalotaxus harringtonia, following the programme of research into novel anticancer agents in the plant kingdom developed by the National Cancer Institute. In fact, the Cephalotaxus alkaloids consist essentially (at least 50%) of cephalotaxine, a biosynthetic precursor of the harringtonines, the latter individually representing only a few percent of the total alkaloids.
Besides their low concentration in the natural state in plant starting material, harringtonines are mixed with many congeners which have very similar chemical structures. Thus, in a high resolution high performance liquid chromatography (HPLC) chromatogram of a semi-purified alkaloid extract, no less than several tens of cephalotaxine esters are counted.
If we consider that:
on the one hand, harringtonines are generally relatively non-crystallogenic, as is suggested by the flexibility of their side chains, which are generally branched and aliphatic,
on the other hand, these esters, in particular harringtonine and homo-harringtonine, are contaminated with congeners which are themselves biologically active and very difficult to separate out, even by high resolution analytical HPLC,
the current state of the art does not allow these compounds to be produced viably on the industrial scale as regards the purity required for pharmaceutical active principles.
Although biosynthetically similar to the alkaloids of the genus Erythrina, cephalotaxanes are alkaloids which have a unique structure in nature, encountered only in the genus Cephalotaxus, which is the only genus of the Cephalotaxacea family. On the other hand, the side chains of the various harringtonine congeners are all derived from the methyl hemiester of the primary carboxyl of (2R) citramalic acid 3a (see Scheme 1 attached) by substitution of the tertiary methyl using alkyl or aralkyl radicals which may themselves be unsubstituted or substituted with tertiary hydroxyls, it then being possible for the latter to form a cyclic ether with a tertiary alcohol (anhydro derivatives).
The attached Scheme 1 shows the main examples of harringtonine congeners, which all have significant cytostatic activity to different degrees. None of the artificial analogous cephalotaxine esters synthesized hitherto in the literature has at least the sub-structure 3b (see Scheme 1) and lack significant cytostatic activity.
It is worthwhile pointing out that, although botanically very similar to the Cephalotaxaceas, Taxaceas contain triterpene alkaloids (taxines), accompanied by non-alkaloid triterpenes, taxanes, which are also of unique structure in nature. Although they are completely different from taxanes in terms of chemical structures and anticancer mechanism of activity, the harringtonines have analogy with taxanes in more than one respect:
they have cytostatic properties,
they consist of a polycyclic skeleton, an inactive biosynthetic precursor of the complete structure, onto which is grafted a side chain containing a similar combination of hydrophilic and hydrophobic substituents,
the polycyclic part of the taxanes (baccatins in the broad sense) and of the harringtonines (cephalotaxines) is relatively abundant in renewable parts of the plant, whereas the active molecules (harringtonines and taxanes) are ten to one hundred times less abundant therein,
the plum yew (Cephalotaxus) is a rare tree, even rarer than the yew (Taxus), and is much less ubiquitous than the latter.
It results from the above facts that, following the manner of the semi-synthesis of taxanes by adding a synthetic chain to a 10-deacetylbaccatin III of extracted origin, the asymmetric semi-synthesis of harringtonines by esterification of a cephalotaxine of natural origin is of considerable medical and economic value. Furthermore, the current population of Cephalotaxus is relatively reduced even in their original habitat. Thus, during its importation into Europe for ornamental purposes last century, Cephalotaxus harringtonia was already no longer present in spontaneous form in eastern China and in northern Japan. The use of a precursor present in a renewable part of the tree (the leaf) in order to prepare homo-harringtonine semi-synthetically is thus of considerable environmental interest, all the more so since the total synthesis of optically active cephalotaxine has not been achieved hitherto, despite the extensive synthetic studies carried out in this respect (a certain number of laborious syntheses of racemic cephalotaxine containing 10 to 15 steps have, however, been carried out: see bibliographic review above).
Consider that several hundred tonnes per year of this rare and very slow-growing tree (even slower growing than Taxus sp.) need to be extracted to satisfy the current market needs for homo-harringtonine (several kilograms per year), whereas the semi-synthesis would consume only a few tonnes of renewable parts of the tree (leaves). Furthermore, homo-harringtonine (HHT) of natural origin currently available on the active principles market is contaminated with its congeners, which, on account of their structural similarity, are very difficult to separate, even by xe2x80x9cpreparativexe2x80x9d high performance liquid chromatography.
First of all, it should be noted that since the use of cephalotaxine itself as a source for semi-synthesis has not yet been economically justified, no process for selectively extracting this substance has been described hitherto. Moreover, among the active compounds, only harringtonine and isoharringtonine have been the subject of American patent applications for their preparation by extraction [R. G. Powell et al., U.S. Pat. No. 3,793,454 and U.S. Pat. No. 3,870,727]. Harringtonine has been the subject of a Japanese patent [JP 58-032,880] and deoxyharringtonine has been the subject of an American patent [U.S. Pat. No. 3,959,312]. As regards the preparation of homo-harringtonine itself, it has been the subject of only a few semi-synthetic studies [T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, 199 (1998)], but no patent application has been made regarding a semi-synthesis process or even an extraction process.
Another aspect which gives the present invention an even greater advantage is that cephalotaxine can serve as a springboard for the synthesis of cephalotaxoids and harringtoids which are useful for antitumour (cancerous and non-cancerous tumours), antiparasitic, antifungal, antiviral and antibacterial chemotherapies.
Harringtonines consist of a complex alkaloid polycyclic alcohol (cephalotaxine), esterified with a side chain, in isolation having no more biological activity than cephalotaxine, but essential for the biological activity of the whole. Saponification of the side chain under harsh conditions leads to the cephalotaxine free base and to harringtonic acids. The attachment of the side chains takes place at the end of the biosynthesis. It has been demonstrated that catabolism leading to this reaction could be triggered in vivo under the influence of environmental or physiological stress exerted on the plant [N. E. Delfel, Phytochemistry, 403 (1980)].
Cephalotaxine, the polycyclic part consisting of 5 fused rings, has a novel arrangement which is unique in nature, i.e. a benzodioxoazepine onto which is fused a spiropyrrolidinopentenediol system. Cephalotaxane contains four asymmetric centres: three xe2x80x9casymmetric carbonsxe2x80x9d and a heterocyclic tertiary aminic nitrogen. The only reactive function is a secondary alcohol located in position 3, the methyl enol ether located in position 2 being potentially sensitive to proton attack. The whole forms a pseudohelical structure encaging the hydroxyl in the tube formed by the tetrahydrazepine. The base cephalotaxine readily forms highly crystallogenic stable salts (for example hydrochlorides and perhydrochlorides).
This alkaloid is relatively insensitive to basic media. On the other hand, several authors describe a certain level of sensitivity to acids and to quaternization of the nitrogen with methyl iodide, leading to a racemization by simultaneous inversion of the 3 asymmetric centres and of the nitrogen [D. J. Abraham, R. D. Rosensten and E. L. McGandy, Tetrahedron Letters, 4085 (1969)]. However, a period of several days in solution at pH 1-4 at 20xc2x0 C. leaves this structure intact (personal observation).
This compound and its congeners which are not O-acylated in position 3 are biologically inactive.
All the side chains for harringtonines which have significant biological activity contain in common the 2-alkyl-2-carbomethoxymethyl-2-hydroxyacetyl unit.
The alkyl chain, of variable length, has at the end either branching constituting an isopropyl bearing (harringtonine HT and homo-harringtonine HHT) or not bearing (deoxy-homo-harringtonine DHT) a tertiary alcohol, or a phenyl radical (for example the neoharringtonine series most recently isolated). In the case of the anhydroharringtonines, the chain can be closed by dehydration between its two tertiary alcohols, for example forming a substituted tetrahydropyran ring. The tertiary carboxyl of this complex diester is borne by the single hydroxyl of the cephalotaxine. The only chiral centre on the side chain is located (to the ester junction. It contains, besides the first to secondary chain, a hydroxyl which, on account of its tertiary nature, does not have the possibility of epimerizing.
Scheme 2 attached shows synthetically the known processes for preparing harringtonines.
Several semi-syntheses of natural cephalotaxine esters and several series of analogues, which have simplified chains but give these analogues reduced cytotoxic activity, have been described hitherto, in particular those of deoxyharringtonine and of isoharringtonine. Most of them relate to simpler and less functionalized esters than those constituting HT and HHT, the esters which are most useful in chemotherapy [for example, deoxyharringtonine, isoharringtonine, T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987)].
All the literature from 1972 to the present date [Mikolajczack et al., Tetrahedron, 1995 (1972); T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxane 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3 totally preformed to give a harringtonine 4b, i.e. the conversion 2a+3e(4b as described in the example featured in the scheme below 
Most of the syntheses described hitherto thus involve binding of the secondary side chain xe2x80x94CH2CO2Me, i.e.:
1st) by the Reformatsky reaction between methyl bromoacetate and the carbonyl (real or potential) on the side chain prebound to cephalotaxine, in the presence of zinc, or
2nd) by prior formation of an organolithium reagent.
All the syntheses described thus consist in esterifying cephalotaxine using the (-keto alkanoyl chloride 7 lacking the end hydroxyl and containing neither the secondary chain located (to the tertiary carboxyl, nor the tertiary hydroxyl (to the carboxyl, to give 8 which is then converted into a harringtonine 4a, according to the reaction described below. 
In formula 8, CTXxe2x80x94 represents the cephalotaxyl radical of formula: 
It should be noted that this (-hydroxyalkylation, which at the same time creates the chiral centre on the side chain, has never been achieved asymmetrically.
A few synthetic routes involve an esterification of cephalotaxine with a substituted hemisuccinyl chloride, optionally followed by subsequent introduction of the tertiary hydroxyl(s).
No O-acylation of cephalotaxine, using totally preformed and functionalized chiral chain precursors (to the tertiary carboxyl, has thus been achieved hitherto [T. Hudlicky, L. D. Kwart and J. W. Reed in xe2x80x9cAlkaloid: Chemical and Biological Perspectivesxe2x80x9d (S. W. Pelletier Ed.), Vol. 5, pages 661 to 675 (1987); M. A. Miah, T. Hudlicky and J. Reed in xe2x80x9cThe Alkaloidsxe2x80x9d, Vol. 51, pages 224 to is 236 (1998)].
Consequently, the methods for preparing harringtonines by semi-synthesis, which have been described to date in the existing art, have the following drawbacks:
absence of stereoselectivity,
poor convergence,
mediocre yields,
functionalization and construction of the chain on a rare and expensive substrate,
chiral homo-harringtonine not obtained to date.
Since cephalotaxine is present in nature in partially racemized form [personal observation; Huang et al., Scientia Sinica, Vol. XXIII, 835 (1980)], the processes of the prior art which use a natural cephalotaxine as starting material can only theoretically result in partially racemized harringtonines. The present invention thus has the advantage of obtaining enantiomerically pure harringtonines even from racemic cephalotaxine, since:
1st) the asymmetric centre on the side chain is created prior to the esterification step, i.e. the side chain precursor can be obtained in enantiomerically pure form prior to being attached,
2nd) the diastereoisomers obtained in the case of a racemic cephalotaxine can be separated by chromatography.
The present invention consists in:
esterifying the hindered free alcohol of a cephalotaxine or alternatively the corresponding metal alkoxide, using a chain in the form of a suitably substituted tertiary carboxylic oxacycloalkane acid which is totally preformed both in terms of the skeleton and in terms of the functionalization, in order to prepare anhydro-homo-harringtonic acids by semi-synthesis.
opening the cyclic side chains thus formed in order to obtain the corresponding diols, i.e. the harringtonines (defined above).
describing a new preparation for all of the diastereoisomers of the dihydroxylated side chains of the harringtonines in a dehydrated cyclic form (anhydroharringtonic acids) or in which the two hydroxyl groups are protected together by difunctional protecting groups forming a ring.
resolving all of the harringtonic and anhydroharringtonic acids, in order to couple them separately with the cephalotaxines.
One part of the present invention thus consists in synthesizing, in particular, anhydroharringtonine, harringtonine, anhydro-homo-harringtonine and homo-harringtonine.
The present invention also relates to esterifying cephalotaxines or metal alkoxides thereof with N-alkyl- and N-carbamoyl-2-alkylisoserine.
Following observations and comparative studies carried out in the taxane series, it was found that, despite a steric bulk which is still greater than that for the hydroxyl of the cephalotaxines, the hydroxyl located at position 13 on the taxane skeleton made it possible to receive acylation with a relatively bulky chain such as, for example, an N-benzoylphenylisoserine protected in position 2xe2x80x2 (although the attempts to acylate baccatin protected with a chain bearing an (hydroxyl group protected with a benzoyl group all failed).
An experimental study of acylation with a very bulky chain, such as pivalic acid, demonstrated the impossibility of acylating the hydroxyl located at position 13 of a baccatin protected by the dialkyl carbodiimide method, whereas the same reaction performed on cephalotaxine showed easy coupling of the pivaloyl chain.
It has also been demonstrated, again in the taxane series, that the improvement in the dynamic compactness of the alkanoyl or aralkanoyl chain is by a bifunctional cyclic protection greatly facilitated the coupling. Thus, for example, French patent applications [J. P. Robin et al., FR 95/12739 and FR 95/15557] indicate that the suitably protected, linear N-benzoylphenylisoserines react in several hours at 80xc2x0 C. with formation of epimerization products, the oxazoline or oxazolidine cyclic precursors reacted in less than one hour at 25xc2x0 C.
The use of the same conditions as above with a chain which has undergone a prior dehydrating cyclization, allowed us to acylate the cephalotaxine or its alkoxides in a few hours at room temperature.
The ease of this acylation was all the more surprising since many authors have designed syntheses which have the drawbacks mentioned above, on the basis of the observation of an impossibility of acylating, explained by the steric hindrance at the two sites: the hydroxyl of the cephalotaxine and the tertiary carboxyl of the side chain precursor [Mikolajczack et al., Tetrahedron, 1995 (1972)].
According to a preferred embodiment or process according to the invention, the hydroxyl function of a cephalotaxane is esterified with a 2-carboxyl-2-alkyl-1-xacycloalkane derivative.
The hydroxyl of a cephalotaxane whose skeleton corresponds to the general formula 1 or, more particularly, a secondary alcohol located in position 3 of a cephalotaxine or metal alkoxides thereof, corresponding respectively to the general formulae CTXxe2x80x94Oxe2x80x94H and CTXxe2x80x94Oxe2x80x94M, M being a metal and CTXxe2x80x94 the cephalotaxyl radical defined above, are esterified with the tertiary carboxylic acid function of a substituted cycloether corresponding to the general formula of the type 3k, or alternatively, preferably, with one of its activated forms, isolated or formed in situ, corresponding to the general formula of the type 3l to give 4c, according to the reaction below: 
R5, R6 and R8 are, independently of each other,
a hydrogen,
a linear or branched and/or cyclic, saturated, unsaturated or aromatic, hydrocarbon-based radical, in particular an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, an aryl, a heterocycloalkyl, the said hydrocarbon-based radical bearing or not bearing (a) heteroatom(s), it being possible for R5 and R8 to be linked together to form a ring
an oxygenated ether bearing one of the above radicals.
n is between 0 and 8.
A is a leaving group capable of leaving behind a carbocation, 3l can thus be, in particular: an acid halide, an anhydride, a mixed anhydride or a cyclic anhydride (when R5=xe2x80x94CH2COxe2x80x94).
The free acid of the type 3k or any activated form of the acids of the type 3l can be used to esterify the cephalotaxanes corresponding to the general formula 1 or, for example, the cephalotaxines of the type CTXxe2x80x94OH. Alternatively, the anhydride of 3l can be prepared separately and isolated, and then placed in contact with the alcohol. This is likewise the case for the cyclic anhydrides of the type 3p 
in which n, R6 and R8 have the same meaning as above, and which can be readily prepared from the corresponding diacids in order once again to give 3k by esterification of methanol or alternatively to become attached as above to the alcohol function of a cephalotaxine of the type 2 with, however, a poorer yield than that above, the primary acid function then being methylated conventionally using methanol in the presence of a protonic acid or a Lewis acid, or alternatively using the boron trifluoride etherate/methanol complex or diazomethane.
Although less effective and more laborious, the method using the acid chloride 3k gave the desired ester 4c.
All the reagents of the type 3k, 3l and 2 as well as the resulting esters of the type 4c, can be used alone in enantiomerically pure form, or in the form of a racemic mixture or in the form of diastereoisomeric mixtures. The intermediates can, in certain cases, not be isolated or formed in situ fleetingly.
The reaction can take place at between 0xc2x0 C. and 140xc2x0 C., with or without an organic solvent, it being possible for these solvents to be alone or as a mixture.
The esterification of the hydroxyl of cephalotaxane with a 2-carboxyl-2-alkyl-1-oxacycloalkane derivative can be carried out either by acyl transfer to the alcohol or by the carbodiimide method.
The esterification reaction by acyl transfer to the alcohol is advantageously carried out according to six specific modes:
(a) esterification of the free acid with the alcohol in acid catalysis,
(b) esterification by acyl transfer via anhydrides or halides,
(c) esterification by acyl transfer using activated esters,
(d) esterification with scandium triflate,
(e) esterification with boron trifluoride etherate,
(f) esterification by the thioester method.
The esterification (a) takes place by placing the acid of the type 3k and the alcohol of the type 2 in contact in solution in a co-solvent and in the presence of an acid catalyst. The displacement of the equilibrium can be promoted by adding a dehydrating agent or by azeotropic entrainment or by partition between two immiscible solvents, one of which is miscible with the ester formed and the other with water. These various methods can be combined.
The acid catalyst can be a protonic acid such as, for example, sulphuric acid, hydrochloric acid, boric acid, preferably para-toluenesulphonic acid, or a Lewis acid which may or may not be supported on a polymer, such as, for example, aluminium chloride, chlorotrimethylsilane or, preferably, boron trifluoride etherate. Advantageously, an ion-exchange resin or bisulphate on graphite can also be used, for example.
The dehydrating agent can be, for example, a dehydrating agent consisting of an inorganic salt which is low in water and inert with respect to the reaction, such as magnesium sulphate, sodium sulphate, calcium chloride or, preferably, a molecular sieve.
The esterification (a) uses the same reaction conditions as above. However, in order to result in the formation of the acylium ion characteristic of this method, the ordinary acid catalysts are replaced, for example, with anhydrous sulphuric acid or superacids such as, for example, hydrofluoric acid and its derivatives or antimony pentafluoride.
The esterification (b) consists in using the same physical operating conditions as above, but using 3l, in which A represents a halogen (acid halide), another alkanoyl molecule which is identical (anhydride) or different (mixed anhydride), such as, for example, trifluoroacetyl, 2,4,6-trichlorobenzoyl, formyl, methoxyformyl, sulphonates, phosphates and chlorophosphate.
In a variant of the above method, and in the specific case in which R5=CH2CO2H, a cyclic anhydride of the type 3p can be used 
in which n, R6 and R8 have the same meaning as above, which can be prepared very simply by treating the corresponding diacid with acetic anhydride, for example under the general operating conditions for the preparation of the mixed anhydrides described below.
The catalysis can be acidic, as indicated in the above method, or, preferably, alkaline, for example a tertiary base such as pyridine and/or dimethylaminopyridine (DMAP), pyrrolidinopyridine (PPY), triethylamine, or a stronger base such as a hydride, for example calcium hydride. The solvent can preferably be an aprotic solvent, for example hexane, toluene, dichloromethane, tetrahydrofuran and/or pyridine.
The esterification (c) is a method similar to the esterification (b). These ester preparation methods can also be advantageously used and involve, for example, 1-acyloxy-1,2,3-triazole or formamidinium or silyl ester or 2-acyloxypyridinium intermediates.
The carbonyidiimidazole method, in which an N-acylimidazole intermediate is involved, can also be used.
The carbodiimide method uses a dehydrating coupling agent such as a carbodiimide, for example dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbo-diimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.
The reaction can be catalysed with a tertiary base such as, for example, pyridine and/or dimethylaminopyridine (DMAP), pyrrolidinopyridine (PPY), triethylamine, 4-morpholinopyridine or any other substituted base. N-Hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HO-Su) can also be used, for example.
The molar ratio of 3l relative to 2 can be between 1/1 and 4/1.
The reaction can preferably be carried out under inert gas at a pressure close to atmospheric pressure, preferably at a temperature of between 0xc2x0 C. and about 110xc2x0 C.
The solvents preferably used are organic solvents such as, for example, toluene and/or dichloromethane and/or chloroform and/or tetrahydrofuran and/or pyridine and/or dimethylformamide.
The application of the methods described above, for the substituted carboxylic oxacycloalkanes, to the coupling of their synthetic precursors, the (linear) 1-hydroxy-1-methoxycarbonylmethylalkenecarboxylic acids, made it possible, against all expectation, to synthesize the esters of the type 4c in a single step from ethylenic tertiary (-hydroxy acids of the type 3f instead of the sequence 3f(3k(3l(4c. 
where m is between 0 and 3,
in formula 4c, n, R5, R6, R8 and CTXxe2x80x94 have the same meaning as above.
Indeed, the ethylenic tertiary (-hydroxy acid 3f treated under conditions similar to those above directly gave the cyclic ester of cephalotaxines of the type 4c without isolation of an intermediate. In the case of the method involving a mixed anhydride 3l, the in situ formation of the corresponding (-lactone has been assumed on account of the presence of an infrared band at 1840 cmxe2x88x921.
These elements were confirmed by the formation and isolation of 3l, using 3f only under the activation conditions described above, i.e. to form the mixed anhydride, for example in the presence of 2,4,6-trichlorobenzoyl chloride, or alternatively, for example, in the so-called DCC method mentioned above.
The substituted carboxylic cycloethers of the type 3k, the substituted ethylenic tertiary (-hydroxy acids of the type 3f their activated intermediates, and cyclic anhydrides of the type 3p, can be coupled with the cephalotaxines, either in the racemic series, or, more advantageously, in the optically active series.
In the case of coupling between one of the above types of acid, in the form of racemic mixtures, with a single enantiomer of a cephalotaxine, a relative stereoselectivity has been found due to the chirality and the major steric hindrance of the reaction site, in the sense that the ratio between each of the two diastereoisomers is generally other than 1.
The separation of the two diastereoisomers formed of the type 4c (R5=CH2CO2Me), known as xe2x80x9canhydroharringtoninexe2x80x9d
in which n, R6, R8 and CTXxe2x80x94 have the same meaning as above, can be carried out by preparative chromatography either in a so-called normal phase, for example on native silica gel as stationary phase and a mixture of organic solvents as mobile phase, or, preferably, in a reverse phase, for example an inert silica grafted with apolar groups such as, for example, organosilyl, cyanoalkyl, phenylalkyl, preferably ocatadecylalkylsilane, chains and a mixture of aqueous solvents as mobile phase.
In the case of an enantiospecific coupling, no trace of epimerization is observed on any of the original parts, and the only diastereoisomer obtained can be crystallized. When this diastereoisomer is not crystalline, it is chromatographed by flash chromatography in order to remove the reagent residues, and is then precipitated by addition to a non-solvent, in order to be isolated in the form of amorphous powder.
The opening of the cephalotaxine carboxylate cycloethers of the type 4c to give the halo alcohol 4d is shown in the following scheme. 
In these formulae n, R5, R6, R8 and CTXxe2x80x94 have the same meaning as above, X being a heteroatom such as a halogen.
The cyclic ethers of the type 4c can, in certain examples, have the particular feature of simultaneously containing a methyl enol ether, for example in position 2 of the cephalotaxines. Despite the usual inertia of true cyclic ethers (i.e. non-hemiacetal ethers), the placing in contact of a solution of 4c in an organic solvent, preferably a chlorinated solvent such as, for example, dichloromethane, dichloroethane or chloroform, under controlled conditions, i.e. in the presence, for example, of a dilute hydrohalic acid, preferably hydrobromic acid in acetic acid, at low temperature, or else in the presence of a halo-trialkylsilane or alternatively of a boron trihalide, for example boron tribromide at low temperature in an organic solvent, preferably a chlorinated solvent, such as, for example, dichloromethane, dichloroethane or chloroform, allowed it to be selectively opened to give a halo alcohol of the type 4d such that X=halogen, with a quantitative yield without any appreciable formation of the O-demethylation product, even in the case of cephalotaxines bearing enol ether(s) or other functions sensitive to ether-cleaving agents. In any case, in the event of an accidental demethylation, the enol can easily be selectively remethylated as described in the literature (for example by dissolution in methanol in the presence of para-toluenesulphonic acid).
The halo alcohols of the type 4d such that X=halogen are of great interest as substrates necessarily leading to heteroatomic analogues, on account of the very good reactivity towards the halogen substitution they bear.
Another variant consists in placing the product of type 4c in contact with an aqueous acid optionally in a miscible or immiscible co-solvent. The acid can be, for example, protonic, and in this case it is an organic or inorganic acid, preferably hydrochloric, tetrafluoroboric or formic acid. In this case, the diols of the type 4b can be isolated directly without passing through the halo alcohol stage described above.
The halo alcohols of the type 4d such that X halogen are hydrolysed according to the following scheme: 
in which n, R6, R8, R5, X and CTXxe2x80x94 have the same meaning as above.
The halo alcohols of the type 4d are particularly suitable for controlled hydrolysis by placing in contact with an aqueous inorganic base such as, for example, dilute sodium hydroxide, sodium carbonate or, preferably, barium carbonate, at a temperature of between 0xc2x0 C. and 30xc2x0 C. with stirring, to give the diols of the type 4b.
One variant consists in carrying out the in situ hydrolysis of the intermediate halo alcohols of the type 4d at the end of the reaction, preferably carried out by treating the cyclic ether of the type 4c in dichloromethane in the presence of hydrobromic acid in acetic acid, and by directly adding the water or the hydrolysis solution to the reaction medium while cold and with vigorous stirring.
The process according to the invention is particularly suitable for the preparation of azaharringtonines, nitrogenous analogues of the harringtonines.
In order to demonstrate, for example, the flexibility of use of halo alcohols of the type 4d such that X=halogen as substrates, these materials were, for example, subjected to azidolysis by treatment using, for example, an alkaline azide in a solvent such as, for example, ethanol, methanol or dimethylformamide, which, by hydrogenolysis in solution in an organic solvent such as, for example, an alkanol or a lower ester, lead to the corresponding amino alcohol corresponding to the formula 4f (aminodeoxyharrington-ine). The amine can then be subjected to amidation under the Schotten-Baumann conditions, i.e. in aqueous media in the presence of an inorganic base as catalyst, to give an amido alcohol of the type 4e
such that Z=NHCOR or NHCOAr, R and Ar being as defined above, more specifically alkyl or aryl groups, respectively, which may or may not be substituted. The amine 4f can also be sec-alkylated to give an alkylaminodeoxyharringtonine (Z=NHR or Z=NHAr or Z=NR2 or Z=NHAr2, it being possible for the two radicals R and Ar to be identical or independent), or acylated to give amides (Z=NHCOR or Z=NHCOAr) or carbamates (Z=NHCOOR) derived from the corresponding aminodeoxyharringtonine 4f,
n, R5, R6, R8, R9, X and CTXxe2x80x94 being defined as above.
Alternatively, the cyclic ethers of the type 4c can be suitable for the Ritter reaction in the presence of a nitrile (which can serve as solvent) at a low temperature of between xe2x88x92100xc2x0 C. and +30xc2x0 C. in the presence of an acid such as sulphuric acid, perchloric acid or, preferably, tetrafluoroboric acid, to give an acylaminodeoxyharringtonine (Z=NHCOR or Z=NHCOAr) derived from the corresponding aminodeoxyharringtonine 4f.
The extraction of the cephalotaxines of the type 2 is carried out according to the procedure indicated below.
The cephalotaxines of the type 2 can be prepared according to the methods described in the literature, either by synthesis or by extraction. In the latter case, since no method uses a direct placing in contact of the plant starting material with an aqueous acid, it has been found to be advantageous to describe this in the present invention. The fresh or dry plant starting material is placed in contact for 24 h with an acidified aqueous-organic mixture using a dilute inorganic acid or a weak organic acid, so as to bring the pH to between 1 and 4, preferably 3. The inorganic acid can be, for example, sulphuric acid or hydrochloric acid and the organic acid can be citric acid, lactic acid or tartaric acid, for example; the organic solvent can be, for example, a lower alkanol, a ketone, tetrahydrofuran or any other water-miscible solvent used in extraction by those skilled in the art. The water content is between 20 and 80%, preferably 50%. The solution obtained can be directly chromatographed or basified in order to be counter-extracted, since, in contrast with the methods described in the literature, it contains no chlorophyll and/or plant fat. The counter-extraction using a water-immiscible organic solvent such as a lower ester or, preferably, a lower halogenated hydrocarbon, more particularly dichloromethane, gives a mixture of total alkaloids isolated in the form of a white powder. Several methods for purifying Cephalotaxus alkaloids exist, but none, in particular in reverse phase, is specifically geared towards the purification of cephalotaxines and more particularly of the cephalotaxine of formula 2a.
The present process for purifying cephalotaxine, which forms an integral part of the novel process for the semi-synthesis of harringtonines, thus involves reverse-phase chromatography, which has never been used for this purpose. This reverse-phase chromatography uses as stationary phase, for example, an inert silica grafted with apolar groups such as, for example, organosilyl, cyanoalkyl, phenylalkyl or, preferably, octadecylalkylsilane chains such as those encountered commercially, and a mixture of aqueous solvents as mobile phase, preferably water itself (without organic solvent); the pH is adjusted, i.e. to between 2 and 4, with an inorganic acid such as hydrochloric or phosphoric or sulphuric acid. It is also advantageous to add an additive such as, for example, aqueous ammonia or triethylamine. According to this process, which is economically very advantageous since it avoids the use of organic solvent and allows the reuse of the stationary phase for virtually hundreds of operations, the cephalotaxine is obtained in a quantitative recovery yield and with a purity of greater than 95%.
The above method makes it possible to obtain not only laevorolatory cephalotaxines naturally present in the plant material, but also racemic cephalotaxines also present in the natural state.
The metal alkoxides, corresponding to the general formula 1b (n=1 to 12) in which M is a metal, more particularly an alkali metal such as sodium, potassium or lithium, or a transition metal, for example zirconium, titanium or zinc, can be obtained by metallation of one or more of the hydroxyls in the mono- or polyhydroxycephalotaxanes corresponding to the general formula 1a (x=1 to 12) and in which M is more particularly an alkali metal or alkaline earth metal or any other metal which can conventionally give rise to the formation of an alkoxide. 
This formation of alkoxide is of great value for acylating more readily in this form the hindered hydroxyls of the cephalotaxanes and most particularly for coupling this cephalotaxane with acylating precursors of the side chains, automatically leading to the harringtonines which are the subject of the present invention.
Several methods can be used to metallate the hydroxyl(s) of a cephalotaxane. For example, a metal hydride, an alkylmetal, an amide or, more generally, any agent capable of exchanging or of giving up a metal atom can be used.
The simple placing in contact of a hydroxylated cephalotaxane in organic solution, preferably under an inert gas, with a metal hydride such as, for example, potassium hydride, lithium hydride or, more particularly, sodium hydride, leads to a cephalotaxane metal alkoxide, which can, for example, to serve as an in situ substrate in order to attach, for example, a suitably substituted alkyl, acyl or alkylsilyl group. The organic solvent can be a suitable aprotic solvent such as an ether, more particularly tetrahydrofuran, a liquid aromatic hydrocarbon, preferably toluene or, more generally, any organic solvent which is liquid under the temperature and pressure conditions used and which has no appreciable reactivity towards the reagent. The temperature of the reaction medium can be between xe2x88x9290xc2x0 C. and +30xc2x0 C.
The simple placing in contact of a hydroxylated cephalotaxane in organic by solution, preferably under inert gas, with a metallated hydrocarbon such as, for example, a lithiated hydrocarbon, preferably butyllithium, leads to the same metal alkoxides as those above. The same solvents as above can be used, except that, since the reactivity of the metal hydrocarbons is generally greater than that of the metal hydrides, the temperature is between xe2x88x92100xc2x0 C. and xe2x88x9220xc2x0 C., preferably between xe2x88x9260xc2x0 C. and xe2x88x9280xc2x0 C.
The simple placing in contact of a hydroxylated cephalotaxane in organic solution, preferably under inert gas, with an amide, preferably an alkali metal amide, for example an alkali metal dialkylamide such as lithium dicyclohexylamide or lithium diisopropylamide or alkali metal (lithium, potassium or sodium) bis(trialkylsilyl)amide leads to the same metal alkoxides as above. The same solvents as above can be used. 
As an example and without detracting from the generality of the present invention, the cephalotaxine 2a in solution stirred in tetrahydrofuran at xe2x88x9270xc2x0 C., treated with one equivalent of butyllithium or lithium bis(trimethylsilyl)amide leads, in a few hours, to the lithium alkoxide 2h, which, when trapped in situ with acetic anhydride, gives the 3-O-acetylcephalotaxine 2b.
The preparation of the substituted racemic carboxylic cycloethers of the type 3k is detailed below.
According to a first variant, the preparation of these carboxylic cycloethers can be carried out by cyclization of the substituted ethylenic tertiary (-hydroxy acids of the type 3f according to the following scheme: 
m, R, R5, R6, R8 and A being defined as above.
The substituted carboxylic cycloethers of the type 3k such as, for example, A or B of formulae: 
can be prepared from the substituted ethylenic tertiary (-hydroxy acids of the type 3f, by simple dissolution in an organic solvent in the presence of an acid.
According to a second variant, the preparation of these carboxylic cycloethers can be carried out by cyclization concomitant with the formation of the acylating species. As mentioned above, acids whose tertiary alcohol is free, such as 3f, cyclize spontaneously by the action of a dehydrating agent required for a certain technique for acylating a cephalotaxine alcohol mentioned above and then esterifying the latter to give 4c.
In the absence of an alcoholic substrate to receive it, the ethylenic tertiary alcohol of the 3f type leads, under anhydrous operating conditions, to the isolation of the acylating intermediate 3l, mentioned above, or, by hydrolysis, to the isolation of the acids of the type 3k.
In this case, the procedure used is strictly the one described for coupling involving the formation of an acylating species in situ but in the absence of a substrate of the cephalotaxine type.
According to a third variant, these substituted carboxylic cycloethers of the type 3k can be prepared by deprotecting the tertiary carboxyl of the suitably substituted precursor 3h, 
R5, R8, R6 and n being defined as above and GP representing a protecting group for the acids, with, as a specific case, GP=R.
According to a fourth variant, in the specific case in which R5=CH2CO2R, the suitably substituted carboxylic cycloethers of the type 3k such that R5=CH2CO2R below, 
R8, R6 and n being defined as above,
can be prepared by total saponification of the corresponding diesters 3l such that R5=CH2CO2R, followed by mild selective methylation of the intermediate diacid 3r such that R5=CH2CO2H. 
Incidentally, this process, carried out at room temperature and with rigorous monitoring of the reaction kinetics, leads to selective saponification of the above primary ester; it thus gives access to derivatives of the type 3t such that R=GP below, 
R8, R6, GP and n being defined as above,
which can be coupled with the cephalotaxines using the methods described above in order to ensure the absence of transesterification during the coupling of the tertiary acids which form the subject of the present invention.
According to a fifth variant, the preparation of these carboxylic cycloethers can be carried out by regioselective methanolysis of the corresponding cydic anhydride.
As mentioned above, the diacid 3r leads, by self-dehydration, to the cyclic anhydride 3p, which is a good acylating agent for alcohols, such that by methanolysis 3k is also preferentially obtained such that R5=CH2CO2Me, which constitutes an additional preparation method.
The preparation of the substituted ethylenic tertiary (-hydroxy acids of the type 3f is detailed below.
The substituted ethylenic tertiary (-hydroxy diacids which are the precursors of the monoacids of the type 3f can, like their cyclic analogues of the type 3k above, be obtained:
either by selective deprotection of their precursors of the type 3i 
or, for example, in the case of the diacids of the type 3s such that R5=CH2CO2H, 
by selective methylation of the primary carboxyl in particular, by placing the reactants in prolonged contact in methanolic solution at room temperature or using the boron trifluoride/methanol complex.
The diacids of the type 3s such that R5=CH2CO2H can be obtained by saponification of the corresponding diesters of the type 3q such that R5=CH2CO2Me by placing the latter in contact with an excess of base in an aqueous or aqueous-alcoholic medium, 
m, R, R8 and R6 being defined as above.
The alcohol can be a lower alcohol such as methanol, ethanol or, preferably, isopropanol, and the base can be, for example, an alkali metal or alkaline earth metal base or a rare earth metal hydroxide or aqueous ammonia. When the reaction takes place at a temperature of between 0xc2x0 C. and 30xc2x0 C. for 15 minutes to 1 hour, the regioselective saponification of the primary ester can be obtained without any resulting difficulty. By increasing the temperature to the boiling point of the solvent mixture and/or by lengthening the reaction time, the diacid is obtained in good yield and without formation of by-products.
The diacids of the type 3s such that R5=CH2CO2H can, on account of their crystallogenic properties, then generally be obtained in enantiomerically pure form by successive crystallizations of enantiomerically enriched mixtures until a constant optical rotation is obtained.
The diacids obtained above can then be converted into substituted ethylenic tertiary (-hydroxy acids of the type 3f such that R5=CH2CO2Me by selective mono-esterification of their primary carboxyl, using methanol in the presence of a protonic acid or a Lewis acid or alternatively using the boron trifluoride etherate/methanol complex or diazomethane.
Incidentally, and as for the cyclic analogues of the type 3t such that R5=CH2CO2H, above, this process carried out at room temperature and with rigorous monitoring of the reaction kinetics leads to selective saponification of the above primary ester; it thus gives access to derivatives of the type 3u such that R5=CH2CO2H, which can be coupled to the cephalotaxines using the methods described above in order to ensure the absence of transesterification during the coupling of the tertiary acids which form the subject of the present invention.
The substituted ethylenic tertiary (-hydroxy esters of the type 3g can be prepared according to the scheme outlined below: 
R6, R8, m and R5 having the same meaning as above.
The ethylenic esters of the type 3g can be prepared according to the numerous methods described in the literature for similar cases such as, for example, (-hydroxyalkylation of the corresponding 1-alkyl- or 1-alkenyl-1-keto ester of the type 9.
As an example and without removing anything from the generality of the present invention, the (-hydroxyalkylation of the 1-alkenyl-1-keto ester of the type 9 with the lithium methoxycarbonylmethyl enolate (R5M=MeOCOCH2Li)or of the corresponding organozinc reagent (Rxc3xa9formatsky reaction, in which R5MX=MeOCOCH2ZnBr) leads to the diester 3g such that R5=CH2CO2Me.
The same reactions applied to a chiral ester (R=R*) lead to a mixture of separable diastereoisomers which, after deprotection of the tertiary acid function, each lead to the diastereoisomer of the pair.
Moreover, the (-hydroxyalkylation reaction of the 1-alkyl- or 1-alkenyl-1-keto ester of the type 9, conducted in the presence of a chiral inducing agent such as sparteine or quinine, can give a significant enantiomeric enrichment, which can be further enhanced by fractional crystallization.
The keto esters of the type 9 are themselves conventionally obtained by C-semi-acylation of the carbanion of the corresponding alkyl or alkenyl halides of the type 10 with a dialkyl oxalate.
One of the advantages over the prior art of the synthetic process which forms the subject of the present invention lies in the possibility of coupling an entirely preformed chain with the cephalotaxines. Thus, the preparation of the above anhydroharringtonic acids in enantiomerically pure form 3k is of considerable interest, since the post-coupling creation of the chiral centre in position 2xe2x80x2 of the harringtonines during the attachment of the secondary chain as described in the prior art leads to an epimeric mixture, on the one hand, which is very difficult to separate, and, on the other hand, to a loss of about 50% of the very precious cephalotaxines (not recyclable in a process for manufacturing a medicinal substance using Good Manufacturing Practice).
Several methods have been used to achieve this aim. They all apply both to the cyclic monoacids of the type 3k or to their diacid precursors of the type 3r, and to their ethylenic linear precursors of the type 3f, it being possible for chiral chromatography methods also to be applied to the precursors which have no function capable of engaging a reversible chemical bond with a chiral species (in this instance free acid functions).
According to a first step of the process for the enantiospecific preparation of these acids, an epimeric mixture is formed by combination with a chiral alcohol or amine.
The reactions for esterifying a hindered secondary alcohol function of a cephalotaxine with oxacycloalkanecarboxylic acids of the type 3k above (including those formed in situ from ethylenic tertiary (-hydroxy acids of the type 3f) can also be applied to the esterification of another chiral alcohol in order to convert a racemic mixture, or one with partial enantiomeric enrichment of acids of the type 3k, into a diastereoisomeric mixture on which all of the non-chiral separation methods become applicable. The above methods are also applicable without modification to the amidation of chiral primary or secondary amines.
Thus, when the oxacycloalkanecarboxylic acids of the type 3k or their ethylenic linear precursors are reacted with a chiral alcohol, denoted by R*OH, or an amide Rxe2x80x2*R*NH (it being possible for Rxe2x80x2* to be replaced with a hydrogen), two chemical species are obtained in which the physicochemical properties are distinct (for example NMR, melting point, solubility, chromatographic properties, enzymatic or microbiological attack, etc.). The alcohol or the amide must preferably be hindered and bear their chiral centre at their site of binding with the tertiary carboxyl of the acid of the type 3k. The alcohol can be, for example, menthol, borneol, valinol or, preferably, quinine. The amine can be, for example, ephedrine; more generally, any commercial chiral alcohol or amide can be used. 
n, R5, R6, R8 and A having the same meaning as above, R* having the same meaning as R, but being chiral.
As an example, and without detracting at all from the generality of the present invention, (xe2x88x92)-quinine, which, like the cephalotaxines, is an alkaloid with a sterically hindered secondary alcohol function, reacts with the racemic mixture of the oxacycloalkanecarboxylic acids of the type 3k to give the mixture of the two corresponding epimers 3v such that R5=CH2CO2Me and 3w such that R5=CH2CO2Me: 
The binary mixtures of epimers obtained by the combination with a chiral compound can be separated, for example, by fractional crystallization, by distillation, by counter-current liquid-liquid partition and, given the high added value of these intermediates, by any common preparative chromatography technique, for example normal phase chromatography, exclusion chromatography, preferably in reverse or normal grafted phase. Since these methods are synergistic, they can advantageously be combined in order to improve the diastereoisomeric purity.
As an example and without detracting at all from the generality of the present invention, the mixture of the two epimers 3v (such that n=3; R6=R8=Me; R5=CH2CO2Me) and 3w (such that n=3; R6=R8=Me; R5=CH2CO2Me), cited in the above example, can be separated without difficulty and in quantitative yield using a grafted phase of octadecylsilane type and a methanol/water mobile phase.
The regeneration of the oxacycloalkane-carboxylic acids of the type 3k in enantiomerically pure form can be carried out by total hydrolysis followed by selective remethylation of the primary carboxyl of the suitably selected diastereoisomer (see above sequence 3j (3k) or, when it is an ester bond with an oxygen in the benzyl position (see for example quinine above), by simple hydrogenolysis. In the latter type of case, the drawback of the hydrogenolysis is largely offset by the economy of a step on an expensive product.
As an example and without detracting at all from the generality of the present invention, (xe2x88x92)-quinine (2xe2x80x2R)-anhydroharringtonate 3v (such that n=3; R6=R8=Me; R5=CH2CO2Me) gave a (2R)-anhydroharringtonic acid of the type 3k and dihydrodeoxyquinine which can thus not be recycled, but this is a minor drawback in view of the low cost of this akaloid. Alternatively, the double saponification of 3v followed by selective remethylation gave a product which was entirely identical to the (2R)-anhydroharringtonic acid of the type 3k above.
The enantiomer of non-natural configuration (2S) can, after having undergone the same conversions as its (2R) enantiomer, be exploited, for example, for the purposes of structure-activity relationship studies.
According to a first step of the process for the enantiospecific preparation of these acids, the racemic mixtures are resolved by formation of salts with a chiral basic species.
The racemic mixtures of oxacycloalkane carboxylic acids of the type 3k (including those formed in situ from ethylenic tertiary (-hydroxy acids of the type 3f), can form a salt with a chiral amine by simple placing together in solution in an organic solvent. Although most of the methods described above for separating the esters and amides formed with 3k are applicable (for example chromatography), since the salts formed are generally highly crystallogenic, it is fractional crystallization which is preferably carried out to resolve the acids of the type 3k. The solvents used, alone or as a mixture, can preferably be polar organic solvents which may or may not be combined with water, such as, for example, ketones, alcohols and lower esters. The reaction to form the salt preferably takes place at a temperature of between 0 and 100xc2x0 C. The recrystallization can be carried out by redissolving the salt in a mixture whose solvent power can be adjusted with precision by means of the use of the above combinations of solvents and by varying the temperature according to the standard techniques practised by those skilled in the art. When the diastereoisomeric enrichment is deemed to be sufficient, the salt is decomposed in the presence, for example, of a dilute aqueous acid such as hydrochloric acid or sulphuric acid. The extraction of the enantiomer of the regenerated acid can be carried out using a water-immiscible organic solvent such as, for example, a lower ester.
As an example and without detracting at all from the generality of the present invention, the racemic mixture of (2R or 2S)-anhydroharringtonic acids of the type 3k can be resolved, for example, by placing them in contact with (xe2x88x92)-ephedrine, followed by fractional recrystallization in an ethyl acetate/methanol mixture.
The 2R-anhydroharringtonic acid of the type 3k is then regenerated by placing the purified salt in contact with 2N hydrochloric acid and continuously extracting the acidic aqueous phase with ethyl acetate.
The oxacycloalkanecarbbxylic acids of the type 3k described above can be subjected to preparative chiral chromatography.
The final products are purified by HPLC to give final products for pharmaceutical use.
Despite the performance levels of the modern methods of synthesis, of semi-synthesis and of isolation of natural substances, it is now established in the regulations issued by the health authorities in industrialized countries that impurity levels of greater than one per thousand (0.1% m/m) in a medicinal substance can be detrimental to the patient.
An identification followed by toxicology studies on any toxicologically unknown substance exceeding this threshold is, moreover, systematically demanded, in order to obtain pharmaceutical files for authorization to market the medicinal products.
The diastereoisomeric purity (with, as a specific case, the enantiomeric purity) can, moreover, lead to therapeutic aberrations; for example, it is well known that quinine (see above formula) is an antimalaria agent, whereas one of its diastereoisomers is a cardiac antifibrillant.
In the therapeutic field of the substances forming the subject of the present invention, it is common to encounter multiplications of from 10 to 100 of the active principle or of a side effect by minor changes (involuntary here) in the molecular structures.
Among the methods for achieving this level of purity, industrial high-resolution chromatography occupies a position of choice, its high cost being an argument which carries little weight compared with the very high added value of the sophisticated active principles, the robustness which it gives to the processes and the safety it offers to users.
As an example, and without detracting at all from the generality of the present invention, homo-harringtonine 4b, such that n=3; R6=R8=Me; R5=CH2CO2Me, CTXxe2x80x94=cephalotaxyl, 
R6, R8, R5, n and CTXxe2x80x94 being defined as above,
can be freed of its epimer at the same time as its other related impurities by preparative reversephase chromatography using a grafted reverse phase of octadecylsilane type as stationary phase and a suitably adjusted methanol/water mixture as mobile phase.
This process gives a product whose sum of related impurities is less than 0.5% and for which none of these impurities taken individually exceeds 0.1%.
The present invention concerns a process for the preparation of sidechain-bearing cephalotaxane of the following formula and/or a salt thereof
xcexa9-COxe2x80x94Oxe2x80x94CTX
where
xcexa9 (xe2x80x9comegaxe2x80x9d) is a representative radical of the chain terminal moiety and xe2x80x94COxe2x80x94 is the carbonyl of the ester group bonded to cephalotaxane;
the xcexa9-COxe2x80x94 radical is corresponding:
either to the following substituted heterocycloalkane formula: 
where n is included between 0 and 8;
Z is oxygen, nitrogen or sulfur heteroatom;
R5, R6 and R8 are independently
hydrogen;
hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s); R6 and R8 may be included in a cycle;
oxygen ether bearing one of the former radicals;
or to the following linear alkene formula: 
where m is included between 1 and 8, R5, R6 and R8 are as defined above;
or to the following formula: 
where n, R5, R6 and R8 are as defined above;
Z and Q2 are independently oxygen, nitrogen or sulfur heteroatom;
Q1 is carbon, silicium or phosphorus atom;
R9 and R10 are independently hydrogen, alkoxy, hydrocarbon radical, including or not heteroatom(s), saturated, unsaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl;
R9 and/or R10 having the ability to be null or taken together to make an heteroatom and/or make a multiple bond with Q1, R9 and R11 having the ability to be null to make a multiple bond between the two atoms of carbon bearing them; and
R11 is hydrogen, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl or alkylcarbonyl;
where
xe2x80x94Oxe2x80x94CTX is cephalotaxine moiety of the following formula a salt thereof: 
where p is equal to 1 or 2;
the two types of radicals -xcexa9 and xe2x80x94CTX above-mentioned being bonded with an ester bond xe2x80x94COxe2x80x94Oxe2x80x94
the said process bringing together:
either carboxylic acid with general formula xcexa9-COxe2x80x94OH or a salt thereof;
or an activated form of an acid with general formula xcexa9-COxe2x80x94A or a salt thereof, with xcexa9-CO of the following formula: 
where n, Z, R5, R6 and R8 are as defined above;
where xcexa9-CO of the following formula: 
m is included between 1 and 8, Z, R5, R6 and R8 are as defined above;
where xcexa9-CO of the following formula: 
and where n, Z, Q1, Q2, R5, R6, R8, R9, R10 and R11 are as defined above
A represents:
either cyclic anhydride of the following formula: 
where n, R6 and R8 are as defined above;
the reaction has been completed by methylation of the primary carboxyl thus formed, with:
either a cephalotaxane or a salt thereof, bearing at least a free hydroxyl group, of the formula Hxe2x80x94Oxe2x80x94CTX, where CTX are as defined above;
or a metallic alcoxide of the formula Mxe2x80x94Oxe2x80x94CTX, where CTX are as defined above and M is a metal;
or an activated form of its hydroxyl group of the formula Yxe2x80x94Oxe2x80x94CTX, where xe2x80x94Oxe2x80x94CTX is as defined above and Y is, either a leaving group to allow a negative charge on oxygen atom by cleavage between Yxe2x80x94 and xe2x80x94Oxe2x80x94CTX, or to allow a carbocation by cleavage between Yxe2x80x94Oxe2x80x94 and xe2x80x94CTX;
with the possible presence of one or several reaction additives to form said sidechain-bearing cephalotaxane and/or a salt thereof.
Most preferably, Z is an oxygen atom and the cephalotaxane Hxe2x80x94Oxe2x80x94CTX is a cephalotaxine of the following formula, or a salt thereof: 
where R1, R2, R3 and R4 are independently hydrogen, hydroxyl group or alkoxide.
A cephalotaxane Hxe2x80x94Oxe2x80x94CTX, as defined above, is cephalotaxine, or a salt thereof, where R1 is hydroxyl, R2 is methoxyl, R3 and R4 are hydrogen. 
R5 is preferably hydrogen or xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94Me.
The xcexa9-CO radical is preferably such as n=1 to 4, R6 and R8 are methyl.
The xcexa9-CO radical may be too such as n=1 or 2, R6 is phenyl and R8 is hydrogen.
When R5 is xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94Me, R1=OH, R2=OMe, R3=R4=H, the cephalotaxane is preferably such as n=0, Z is a nitrogen atom and R8 is hydrogen.
A may be xcexa9-COxe2x80x94CO where xcexa9 is defined as above, or an halide. A may also be a radical of compound xcexa9-COxe2x80x94A having the ability to generate cleavage of the bond between carbonyl group and substituent A to provide xcexa9-CO+ and Axe2x88x92.
In addition, A is a radical selected from substituents:
mxc3xa9thoxyformyloxy of formula MeOCOOxe2x80x94,
trifluoroacxc3xa9tyloxy of formula CF3COOxe2x80x94,
alkylsulfonoxy of formula RSO3xe2x80x94,
phosphoxy of formula (RO)2POxe2x80x94,
halophosphoxy of formula ROP(Cl)Oxe2x80x94,
trialkylsilyloxy of formula R3SiOxe2x80x94,
formulas wherein R is alkyl,
dimxc3xa9thyl-formamidinium chloride of formula 
or acyloxy-pyridinium bromide of formula 
A may also be 2,4,6-trichlorobenzoyloxy or a radical corresponding to the following formula: 
In the case where A is a carbonyl-diimidazole, where A is 2,4,6-trichlorobenzoyloxy, the reagent of formula xcexa9-COxe2x80x94A is obtained by contacting an acid xcexa9-COxe2x80x94OH, as defined above, with 2,4,6-trichlorobenzoyloxy carbonyl-diimidazole in presence of a strong base such as an alkoxide.
According carbodiimide method, the coupling additive is a substituted carbodiimide and/or a basic additive such as tertiary amine for example.
For example, the substituted carbodiimide is selected from cyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC) and chlorhydrate of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.
The cephalotaxine alcoxide, corresponding to the formula Mxe2x80x94Oxe2x80x94CTX where M and CTX are as defined above, may be obtained by contacting a cephalotaxine of formula Hxe2x80x94Oxe2x80x94CTX with metal himself, an amidure, a metallic hydride or an alkyl-metal.
M may be an alkaline metal such as lithium, potassium or sodium. The aim of the present invention is also the preparation of new compounds such as:
the lithium alcoxide of cephalotaxine corresponding to the following formula: 
the sodium alcoxide of cephalotaxine corresponding to the following formula: 
a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof: 
xe2x80x83where
n is included between 0 and 8;
Z is oxygen, nitrogen or sulfur heteroatom;
R5, R6 and R8 are independently
hydrogen;
hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl. cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s);
oxygen ether bearing one of the former radicals;
CTX is as defined above;
except for compounds where Z is oxygen atom and,
1xc2x0) n=2 or 3, and simultaneously R6=R8=methyl and R5=OMe or hydroxyl,
1xc2x0) n=2 and simultaneously R6=R8=methyl and R5=OMe or hydroxyl;
3xc2x0) n=3 and simultaneously R6 is hydroxyl, when R8 is mxc3xa9thyl and R5 is xe2x80x94CH2CO2CH3 radical.
a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof: 
xe2x80x83where
m, R5, R6, R8 and CTX are as defined above;
except compound where m=2, R5=CH2CO2CH3, R6=R8=methyl and CTX is as defined above.
R5 is preferably the xe2x80x94CH2xe2x80x94COxe2x80x94Oxe2x80x94CH3 radical.
a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof: 
where n, Z, Q1, Q2, R5, R6, R8, R9, R10, R11 and CTX are as defined above.
Preferably, Q2 is oxygen atom and/or Z is nitrogen atom and the cephalotaxane such as n=0.
a sidechain-bearing cephalotaxane corresponding to the following formula: 
a sidechain-bearing cephalotaxane corresponding to the following formula: 
a sidechain-bearing cephalotaxane corresponding to the following formula: 
When the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula: 
where n, R5, R6, R8, CTX and Z are as defined above, the said chain is open with an agent and/or a protonic or not protonic electrophilic radical E in aqueous or not aqueous medium, to provide an intermediate compound of the following formula: 
where n, CTX, R5, R6 and R8 are as defined above, E is either hydrogen or the provisionally or definitively fixed eletrophilic radical;
the aforementioned intermediate compound may be attacked with an agent or a nucleophilic radical Zxe2x80x2, deliberately added or possibly present in the medium, and
when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula: 
where n, R5, R6, R8, R9, R10 and R11 are as defined above, and Zxe2x80x2 is an heteroatom;
the said chain is open by hydrolysis or carefully solvolysis with possibly presence of activation and/or opening additive.
In addition, to provide an open sidechain-bearing cephalotaxane of the following formula: 
where n, CTX, R5, R6 and R8 are as defined above;
Zxe2x80x2 is:
either a halogen or an heteroatom bearing a hydrogen or a radical R11 such as defined above;
or an hydrogen, hydrocarbon radical, the said radical bearing or not heteroatom(s), saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or heterocycloalkyl.
For example, cephalotaxine esters of the following formulas: 
Where R5, R6, R8, Zxe2x80x2, X and CTX are as defined above;
bromodeoxyharrintonine (n=2) and bromodeoxyhomoharrintonine (n=3) 
where CTX is as defined above;
aminodeoxyharrintonine (n=2) and aminodeoxyhomoharrintonine (n=3) 
where CTX is as defined above;
In addition, when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula: 
where n, R5, R6, R8, CTX and Z are as defined above, the said chain is open by treatment with a solution of hydrobromic acid in acetic acid, in an halogenated solvent, preferably dichloromethane, followed by in situ hydrolysis to provide, without isolation of the intermediate, a sidechain-bearing cephalotaxane of the following formula: 
where n, CTX, R5, R6 et R8 are defined above.
Acids corresponding to the following formula:
xcexa9-COxe2x80x94OH
where xcexa9 radical is as defined according above;
the said formula equivalent to racemic mixture containing compounds of the formulas (+)-xcexa9-COxe2x80x94OH and (xe2x88x92)-xcexa9-COxe2x80x94OH such as (+)-xcexa9-COxe2x80x94OH represents its dextrogyre enantiomer and (xe2x88x92)-xcexa9-COxe2x80x94OH represent its levogyre enantiomer, were obtained
a) by contacting of said racemic mixture or one of its activated form of the formula
xcexa9-COxe2x80x94A
xe2x80x83which is as defined above;
the said racemic mixture or said activated form generating respectively:
either an anion corresponding to the formula (xcexa9-COxe2x80x94O)xe2x88x92;
or a cation corresponding to the formula (xcexa9-CO)+;
with a pure enantiomeric form of chiral entity, said xe2x80x9cresolution agentxe2x80x9d symbolized by xcex94*(delta stella), having the ability to form:
either a stable combination, by covalent bonding;
or an easily reversible labil combination, by hydrogen bonding or by hydrophobic interaction;
or intermediate lability bonding by electrostatic interaction;
to provide a diastereomeric mixture of xcexa9-COxe2x80x94O-xcex94* and de xcexa9-CO-xcex94*;
b) then by physical separation of the mixture of two diastereomers or two complex compounds or more generally of two new entities physically and/or chemically different then obtained;
c) then by regeneration and finally separation of each one of enantiomers of the generic formula xcexa9*-COxe2x80x94OH, where xcexa9*( less than  less than omxc3xa9ga stellar greater than  greater than ) represents the generic symbol of the same chiral radical in the either one or the other pure enantiomeric forms corresponding to the following formulas (+)-xcexa9-COxe2x80x94OH and (xe2x88x92)-xcexa9-COxe2x80x94OH which are as defined above.
Preferably, xcexa9-COxe2x80x94 is:
either a radical corresponding to the following formula: 
where n, Z, R6, R8, and R5 are as defined above;
or a radical corresponding to the following formula: 
where m, Z, R6, R8, and R5 are as defined above;
or a radical corresponding to the following formula: 
where n, R5, R6, R8, Z, Q2, Q1, R9, R10 and R11 are as defined above.
The stable combination may be represented by an ester of the following formula xcexa9-COxe2x80x94O-xcex94* such as xcexa9 and xcex94* are as defined above, the said stable combination is obtained by contacting acid with a chiral alcohol corresponding to the formula HO-xcex94* such as xcex94* is as defined above, according the process of invention.
The stable combination may be represented by an amide corresponding to the either one or the other formulas xcexa9-COxe2x80x94NH-xcex94* or xcexa9-COxe2x80x94N-xcex94* such as xcexa9 and xcex94* are as defined above, the said stable combination is obtained by contacting acid with primary or secondary chiral amine corresponding to formulas H2N-xcex94* or NN=xcex94* such as xcex94* is as defined above, according the process of the invention.
The stable combination may be represented by an thioester of the following formula xcexa9-COxe2x80x94S-xcex94* such as xcexa9 and xcex94* are as defined above, the said stable combination is obtained by contacting acid with a chiral thiol corresponding to the formula HS-xcex94* such as xcex94* is as defined above, according the process invention.
Finally, the ionic combination may be represented by a salt just prepared by contacting of acid with a chiral amine corresponding to the either one or the other of the three following formulas:
xcexa9-COxe2x80x94Oxe2x88x92[NH-xcex94*]+
xcexa9-COxe2x80x94Oxe2x88x92[NH2-xcex94*]+
xcexa9-COxe2x80x94Oxe2x88x92[NH3-xcex94*]+
where xcexa9 and xcex94* are as defined above.
The bringing into play of a labil bonding based combination is achieved in the form of chromatography with the help of a chiral stationary phase.
The bringing into play of an interatomic or intermolecular labil bonding based combination, within crystalline latice, is achieved in the form of fractionated crystallization initiated by a chiral precursor.
The chiral alcohol HO-xcex94* is:
either (xe2x88x92)-quinine corresponding to the following formula: 
or (xe2x88x92)- or (+)-methyl mandelate corresponding to the following formulas: 
or (xe2x88x92)- or (+)-menthol corresponding to the following formulas: 
The chiral amine H2N-xcex94* is (xe2x88x92)- or (+)-ephedrine corresponding to the following formulas: 
The present invention concerns the following new compounds:
the (xe2x88x92)-quinidyl (2xe2x80x2R)-(xe2x88x92)-anhydro-homoharringtonate and the (xe2x88x92)-quinidyl (2xe2x80x2S)-(xe2x88x92)-anhydro-homoharringtonate corresponding respectively to the two following formulas: 
the (xe2x88x92)-menthyl (2xe2x80x2R)-(xe2x88x92)-anhydro-homoharringtonate and the (xe2x88x92)menthyl (2xe2x80x2S)-(xe2x88x92)-anhydro-homoharringtonate corresponding respectively to the two following formulas: 
the (xe2x88x92)-methylmandelyl (2xe2x80x2R)-(xe2x88x92)-anhydro-homoherringtonate and the (xe2x88x92)-methylmandelyl (2xe2x80x2S)-(xe2x88x92)-anhydro-homoharringtonate corresponding respectively to the two following formulas: 
the (xe2x88x92)-ephedrinium (2xe2x80x2R)-(xe2x88x92)-anhydro-homoharringtonate and the (xe2x88x92)-ephedrinium (2xe2x80x2S)-(xe2x88x92)-anhydro-homoharringtonate corresponding respectively to the two following formulas: 
According the process of invention, when the carboxylic acid is the tertiary heterocycloalcane carboxylic acid corresponding to the following formula: 
where n, Z, R5, R6 and R8 are as defined above, the said acid is obtained by treatment in aprotic or protic solvant, eventually in the presence of cyclization additive and/or dehydrating agent, the said treatment eventually supported with physical carrying of the water formed.
or open tertiary ethylenic acid corresponding to the following formula: 
where m, Z, R5, R6 and R8 are as defined above.
or open tertiary ethylenic acid corresponding to the following formula: 
where m is included between 1 and 8, Z, R5, R6 and R8 are as defined above,
R12 is not a CTX radical as defined above and represents R5 and/or a protective group of acids and/or a chiral group;
then R12 is removed later, either just by saponification, or by hydrogenolysis, or more generally by the method of the state of art to remove protective groups of acids.
In the absence of cyclization additive, the reaction of cyclization just take place by heating.
Preferably, the cyclization additive is a protic acid such as sulfonic or formic acid, or an aprotic acid, included in immobilized form.
In the step of preparation of the acid described above, Z is an oxygen atom.
The aim of the present invention is also the preparation of the following new compounds:
the tertiary heterocycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n, Z, R5, R6 and R8 are as defined above, and R5 is not hydrogen;
except for compounds where Z is oxygen atom and,
1xc2x0) n=0 and R5 is not xe2x80x94CH2CO2H or xe2x80x94CH2CO2CH3 radical;
2xc2x0) n=0 and R5 is xe2x80x94CH2CO2H or xe2x80x94CH2CO2CH3 radical, and R6=R8=methyl or xe2x80x94CH2CO2H or xe2x80x94CH2CO2CH3 radical;
3xc2x0) n=2 and simultaneously R6=R8=methyl, and R5=OMe or hydroxyl;
4xc2x0) n=2 and simultaneously R6=R8=methyl, and R5 is xe2x80x94CH2CO2H or xe2x80x94CH2CO2CH3 radical or methyl;
5xc2x0) n=3 and simultaneously R6 is hydroxyl, and R8 is methyl, and R5 is xe2x80x94CH2CO2CH3 radical;
6xc2x0) n=3 and simultaneously R6=R8=methyl and R5=OH or methyl or ethyl.
the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 0 and 8, R5, R6 and R8 are as defined above, but are not hydrogen simultaneously.
except for compounds corresponding to the exceptions 1 to 6 defined above.
the tertiary heterocycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 0 and 8, Z, R5, R6 and R8 are as defined above, R5 is not hydrogen, and R12 is not a CTX radical defined above;
except for compounds where Z is oxygen atom and, corresponding to the exceptions 1 to 6 defined above.
the tertiary oxacycloalcane carboxylic hemiester, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 0 and 8, R6 and R8 are as defined above.
except for compounds corresponding to the exceptions 1 to 6 defined above.
the tertiary oxacycloalcane carboxylic hemiester, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 0 and 8, R6 and R8 are as defined above, R12 is an hydrocarbon radical different from CTX as defined above.
except for compounds corresponding to the exceptions 1 to 6 defined above.
the tertiary oxacycloalcane carboxylic hemiester or anhydro-homoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary oxacycloalcane carboxylic hemiester or anhydro-harringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n and R5 are as defined above, except for compounds corresponding to the exceptions 1 to 6 defined above.
the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 1 and 8.
the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where n is included between 0 and 8.
the tertiary oxacycloalcane carboxylic acid or oxanhydroneoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary oxacycloalcane carboxylic acid or oxanhydro-neohomoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where m is included between 1 and 8, R6 and R8 are as defined above, but are not hydrogen simultaneously, and R5 is not hydrogen or heteroatom.
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where m, R6, R8 and R12 are as defined above, and mxe2x80x2 is included between 1 and 8.
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where m is included between 1 and 8, R6 and R8 are as defined above but are not hydrogen.
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula: 
where m is included between 1 to 8, preferably m=1.
the anhydrides of acid of the general formula xcexa9-COxe2x80x94Oxe2x80x94CO-xcexa9 where xcexa9 is as defined above.
the mixed anhydrides of acid of the general formula Oxe2x80x94COxe2x80x94A where A is a radical selected from the following substituents:
mxc3xa9thoxyformyloxy of formula MeOCOOxe2x80x94,
trifluoroacxc3xa9tyloxy of formula CF3COOxe2x80x94,
alkylsulfonoxy of formula RSO3xe2x80x94,
phosphoxy of formula (RO)2POxe2x80x94,
halophosphoxy of formula ROP(Cl)Oxe2x80x94,
trialkylsilyloxy of formula R3 SiOxe2x80x94,
formulas wherein R is alkyl.
dimxc3xa9thyl-formamidinium chloride of formula 
acyloxy-pyridinium bromide of formula 
and 2,4,6-trichlorobenzoyloxy.
the mixed anhydride corresponding to the following formula: 
the acid chlorides defined above, corresponding to the general formula xcexa9-COxe2x80x94X, where X is halogen.
the cyclic anhydrides corresponding to the following formula: 
where n, R6 and R8 are as defined above.
the cyclic anhydride corresponding to the following formula: 
In the process according to the invention, the sidechain-bearing cephalotaxane was purified like a salt by chromatography using a hydrophobic reversed-phase like stationary phase, and a mobile phase without organic solvent like a solution adjusted to a pH 2 to 4.5 with a buffer prepared with an acid and an alkaline or ammonium salt and one or several additive like attenuator of silanol effect, the said cephalotaxine salt was generated from mineral acid under the form of chlorohydrate, sulfate, phosphate, nitrate, perchlorate, or from organic acid under the form of tartrate, malate, citrate or lactate.
In the process according to the invention, the sidechain-bearing cephalotaxane was purified by a step of chromatographic purification of a natural or semi-synthetic or synthetic homoharringtonine as a pharmaceutical use corresponding to the following formula: 
to remove the undesired related impurity named 2xe2x80x2-xc3xa9pi-homoharringtonine resulting:
a) either from a semi-synthetic process with introduction of a synthetic homoharringtonic acid of inadequate enantiomeric purity, the generated impurity showing the absolute configuration corresponding to the following 
b) or from the biosynthetic process in the plant, where a cephalotaxine with inadequate enantiomeric purity was introduced, or in the form of artefact by partial racemization of the cephalotaxine moiety, the generated impurity showing strictly identical chromatographic properties with a non-chiral system, with an absolute configuration opposite to the one above (enantiomer) and corresponding to the following formula: 
especially making use of one of the following chromatographic systems:
A) Stationary phase: alkyl- or phenyl- or alkylphenyl- ou phenylalkyl-silane, preferably n-octadecylsilane,
B) Mobile phase: water-tetrahydrofurane, water-methanol, water-acetonitrile or buffer pH 2 to 6.5 in replacement of water, or all other mobile phase with equivalent selectivity,
This process of purification and chromatographic control of a natural or semi-synthetic or synthetic homoharringtonine, allows to offset the double insufficiency of enantiomeric purity of the semi-precursors, both on the sidechain precursor (said homoharringtonic acid) and cephalotaxine, the two said-precursors are each independently generated by total synthesis or semi-synthetic process or natural process within of the plant (biosynthesis), in fact the withdrawal of the non natural enantiomer of homoharrintonine showing an opposite absolute configuration, by using a chiral stationary phase with preparative scale.
The present invention is illustrated without implied limitation by the following schemes.
Scheme 1 in annex gives the definition and the formulas of main harringtonines.
Scheme 2 recapitulates process of preparation of harringtonines of prior art.
Scheme 3 gives the sequence of synthesis of homoharringtonine corresponding to the Example 25, where A represents a 2,4,6-trichloro-phxc3xa9nyl group, R represents a methyl and Rxe2x80x2 represents a cephalotaxyl moiety.
Scheme 4 represents a variant of the process according to the invention, more exactly the semi-synthesis of harringtonines via oxacycloalcalne carboxylic acids. The substitutes R6, R7, R8, R, A CTX, X and the letters n et m referenced in this scheme are defined in the description.
The following definitions applies for the whole present document.
Alkaloids: natural substances present in the vegetal kingdom allowing at least a cyclic or acyclic basic nitrogen (allowed extensions: animal kingdom, primary amine; refused extensions: amidic nitrogen, because non basic, ex: taxanes) and showing frequently pronounced pharmacological properties.
Basic or free Alkaloid: alkaloid showing a tertiary amine in a non-ionized form generally existing at alkaline pH and soluble in aprotic organic solvents.
Salt of alkaloid or just  less than  less than salt greater than  greater than : ionized form of alkaloid with amine function showing a positive formal charge and a negative counter-ion, actually more soluble in water and protic solvents and less soluble in aprotic solvents.
Cephalotaxanes 1 (see scheme 1): this generic term indicates the basic framework, showing diverse oxygenated substitutes (aliphatic or aromatic ether, free or esterified alcohol, enol and/or free or substituted phenol, bridged ether, more generally all substitute usually founded at natural state for this kind of compounds).
Cephalotaxines 2: this generic term indicates cephalotaxanes possibly showing at least one of the substitutes described above, a sidechain excepted.
Cephalotaxine 2: a cephalotaxine in majority present in the genus Cephalotaxus 
Cephalotaxoids: this generic term indicates a non-natural cephalotaxine.
Harrinatonines: this generic term indicates cephalotaxane showing at least an alcohol group, a phenol or an enol, esterified by a sidechain and possibly by one of the substitutes described above.
Harringtonine (the): one of the main alkaloids bearing a sidechain in position 3.
Harrinqtoids: this generic term indicates a non-natural harringtonine, where sidechain is an ester radical showing at least 3 carbon atoms.
Sidechain of the harringtoids: this generic term indicates an ester formed between one of the hydroxyl group and a carboxylic acid showing at least 3 carbon atoms allowing usually a tertiary alcohol tertiaire in a position and an hydrophobe substitute in xcfx89 position relatively to the carboxyl group.
Adaptation of the additive et subtractive empirical nomenclature suitable for cxc3xa9phalotaxanes.
Prefixes of common nomenclature are usually used in the literature to indicate structural variations of the sidechain of harringtonines (see Examples in the table of scheme 1). The sidechain of reference is this of harringtonine showed in formula 3b, n=2, R=H, R7=H, R6=2-hydroxyisopropyl.
Homo: 1 extra carbon.
Bishomo: 2 extra carbons.
Nor: a sidechain with 1 carbon less.
Iso: a sidechain with methylene bearing an hydroxyl group at the place of juxtaterminal carbon.
Deoxy: the hydroxyl group of juxtaterminal carbon is replaced par hydrogen.
Anhydro: the two tertiary hydroxyl groups lose a molecule of water to give the corresponding saturated oxygenated heterocycle.
neo: R6 is a phenyl group at the place of 2-hydroxyisopropyl.
HPLC: High-Performance Liquid Chromatography.
NMR: Nuclear Magnetic Resonance.